ABSTRACT Cardiac myosin binding protein-C (cMyBP-C) is an essential regulator of heart function that is necessary for normal systolic and diastolic heart function and for enhanced contractility in response to inotropic agonists that phosphorylate cMyBP-C through numerous signaling pathways. Whereas phosphorylation is associated with cardiac protection, hypo-phosphorylation is consistently found in heart failure. Mutations in MYBPC3, the gene encoding cMyBP-C, are also the most common cause of hypertrophic cardiomyopathy (HCM), a disease with a prevalence of ~1:500. However, despite its significance to cardiac health and disease, there is still remarkably little known regarding how cMyBP-C mediates its functional effects or how cMyBP-C phosphorylation increases cardiac contractility in response to inotropic stimuli. Work from our 3 labs has shown unequivocally that cMyBP-C directly activates the thin filament in muscle sarcomeres in the same way as myosin cross-bridges. Our data thus demonstrate cMyBP-C effects to augment contraction or delay relaxation are due to direct effects of cMyBP-C to shift tropomyosin to an activated state on the thin filament. Here we take advantage of the revolution in cryo-EM to reveal detailed structures of cMyBP-C interactions with the thin filament and also with myosin-S1 for the first time. We will use cryo-EM reconstructions to identify key residues that mediate interactions with thin filaments and myosin-S1 and will test model predictions using an arsenal of functional approaches including a novel (?Spy-C?) method developed exclusively in our labs that allow us to replace the N'-terminal domains of cMyBP-C in sarcomeres in situ. Using our structural maps, we demonstrate proof of principle by identifying amino acids responsible for cMyBP-C activation of tropomyosin and show that phosphorylation causes cMyBP-C domains to reposition on the thin filament. Specific aims will build on these discoveries and combine our unique resources to: 1) Determine how individual cMyBP-C domains communicate with neighboring domains when bound to the thin filament and myosin-S1, and 2) Define how Ca2+ and phosphorylation modulate key regulatory interactions of cMyBP-C with the thin filament and myosin- S1. By identifying the critical molecular interactions that control cMyBP-C binding to the thin filament and myosin-S1 these studies will pave the way for future targeted approaches to modulate cardiac contraction and relaxation.